Control mechanisms are of crucial importance for the maintenance of normal metabolism and a knowledge of the molecular details of the mechanisms that regulate metabolism is essential for elucidation of pathological processes. Thus, knowledge of the molecular mechanisms for regulation of the urea cycle, the major pathway of ammonia removal, will provide an understanding of disorders (e.g., hepatitis, cirrhosis, ana metabolic defects) in which liver function is temporarily or permanently altered. Carbamoyl phosphate synthetase (CPSase), which catalyzes the entry step of the urea cycle is known to be the primary site of control. However, the molecular mechanisms for this control have not been elucidated. Previous studies have established that CPSase utilizes two molecules of ATP at two discrete sites on the enzyme and that the enzyme is composed of four structural domains (A-D), with one ATP site involving peptides from domains B and D, the other ATP site involving peptides from domains C and D, and the site for the required effector N-acetylglutamate involving peptides from domains A, B, and D. The proposed research is aimed at the following: further definition of the regions/residues of domains B, C, and D which are essential for ATP binding and/or catalysis, by construction of deletions and site-directed mutations in these domains; further elucidation of the structural basis for the required N-acetylglutamate activation of CPSase, by construction of deletions, site-directed mutations and chimeric enzymes; definition of the site for ammonia binding on CPSase, by screening for mutant growth requirements, followed by purification and kinetic analysis; further elucidation of the basis for the interaction of CPSase with the mitochondrial membrane, by determination of the mechanism for cardiolipin interaction with the enzyme; and, definition of the monomeric and oligomeric enzyme configurations, by utilization of electron microscopy and comparison to solved structures of other proteins. The overall goal of the proposed research is to develop a detailed model of the CPSase structure and its role in enzymatic function.